Extracellular Vesicles of Patients on Peritoneal Dialysis Inhibit the TGF-ß- and PDGF-B-Mediated Fibrotic Processes.

Among patients on peritoneal dialysis (PD), 50-80% will develop peritoneal fibrosis, and 0.5-4.4% will develop life-threatening encapsulating peritoneal sclerosis (EPS). Here, we investigated the role of extracellular vesicles (EVs) on the TGF-ß- and PDGF-B-driven processes of peritoneal fibrosis. EVs were isolated from the peritoneal dialysis effluent (PDE) of children receiving continuous ambulatory PD. The impact of PDE-EVs on the epithelial-mesenchymal transition (EMT) and collagen production of the peritoneal mesothelial cells and fibroblasts were investigated in vitro and in vivo in the chlorhexidine digluconate (CG)-induced mice model of peritoneal fibrosis. PDE-EVs showed spherical morphology in the 100 nm size range, and their spectral features, CD63, and annexin positivity were characteristic of EVs. PDE-EVs penetrated into the peritoneal mesothelial cells and fibroblasts and reduced their PDE- or PDGF-B-induced proliferation. Furthermore, PDE-EVs inhibited the PDE- or TGF-ß-induced EMT and collagen production of the investigated cell types. PDE-EVs contributed to the mesothelial layer integrity and decreased the submesothelial thickening of CG-treated mice. We demonstrated that PDE-EVs significantly inhibit the PDGF-B- or TGF-ß-induced fibrotic processes in vitro and in vivo, suggesting that EVs may contribute to new therapeutic strategies to treat peritoneal fibrosis and other fibroproliferative diseases.

In situ captured antibacterial action of membrane-incising peptide lamellae.

Developing unique mechanisms of action are essential to combat the growing issue of antimicrobial resistance. Supramolecular assemblies combining the improved biostability of non-natural compounds with the complex membrane-attacking mechanisms of natural peptides are promising alternatives to conventional antibiotics. However, for such compounds the direct visual insight on antibacterial action is still lacking. Here we employ a design strategy focusing on an inducible assembly mechanism and utilized electron microscopy (EM) to follow the formation of supramolecular structures of lysine-rich heterochiral ß3-peptides, termed lamellin-2K and lamellin-3K, triggered by bacterial cell surface lipopolysaccharides. Combined molecular dynamics simulations, EM and bacterial assays confirmed that the phosphate-induced conformational change on these lamellins led to the formation of striped lamellae capable of incising the cell envelope of Gram-negative bacteria thereby exerting antibacterial activity. Our findings also provide a mechanistic link for membrane-targeting agents depicting the antibiotic mechanism derived from the in-situ formation of active supramolecules.

Molecular imaging of bacterial outer membrane vesicles based on bacterial surface display.

The important roles of bacterial outer membrane vesicles (OMVs) in various diseases and their emergence as a promising platform for vaccine development and targeted drug delivery necessitates the development of imaging techniques suitable for quantifying their biodistribution with high precision. To address this requirement, we aimed to develop an OMV specific radiolabeling technique for positron emission tomography (PET). A novel bacterial strain (E. coli BL21(DE3) ΔnlpI, ΔlpxM) was created for efficient OMV production, and OMVs were characterized using various methods. SpyCatcher was anchored to the OMV outer membrane using autotransporter-based surface display systems. Synthetic SpyTag-NODAGA conjugates were tested for OMV surface binding and 64Cu labeling efficiency. The final labeling protocol shows a radiochemical purity of 100% with a ~ 29% radiolabeling efficiency and excellent serum stability. The in vivo biodistribution of OMVs labeled with 64Cu was determined in mice using PET/MRI imaging which revealed that the biodistribution of radiolabeled OMVs in mice is characteristic of previously reported data with the highest organ uptakes corresponding to the liver and spleen 3, 6, and 12 h following intravenous administration. This novel method can serve as a basis for a general OMV radiolabeling scheme and could be used in vaccine- and drug-carrier development based on bioengineered OMVs.

Dual-Role Peptide with Capping and Cleavage Site Motifs in Nanoparticle-Based One-Pot Colorimetric and Electrochemical Protease Assay.

A new method for enzyme substrate assembly and its use in proteolytic enzyme assays with colorimetric and electrochemical detection is presented. The novelty of the method is the use of dual-function synthetic peptide containing both gold clustering and protease-sensitive moieties, which not only induces the simple formation of the peptide-decorated gold nanoparticle test substrates but also allows for the detection of proteolysis in the same batch. Protease-treated nanoparticles with a destabilized peptide shell became more prone to electroactivity, and thus, the model enzyme plasmin activity could be quantified with stripping square wave voltammetry analysis as well, giving an alternative method to conduct aggregation-based assays. Spectrophotometric and electrochemical calibration data proved to be linear within the 40-100 nM active enzyme concentration range, with possible extensions of the dynamic range by varying substrate concentration. The simple initial components and the ease of synthesis make the assay substrate preparation economic and easy to implement. The possibility of cross-check analytical results with two independent measurement techniques in the same batch greatly increases the applicability of the proposed system.

Structuring liquids through solvent-assisted interfacial association of oppositely charged polyelectrolytes and amphiphiles.

HYPOTHESIS: Sculpting liquids into different shapes is usually based on the interfacial interactions of functionalized nanoparticles or polymers with specific ligands, leading to exciting material properties due to the combination of the mobility of liquid components with the solid-like characteristic of the arrested liquid/liquid interface. There is an intense interest in novel structured liquids produced from simple compounds with versatile application potentials. Complexes of oppositely charged commercial polyelectrolytes and traditional aliphatic surfactants are good candidates for this goal since they reveal rich structural features and could adsorb at various interfaces. However, they have not been applied yet for structuring liquids. EXPERIMENTS: The interfacial interactions and film formation between aqueous sodium poly(styrene) sulfonate solutions (NaPSS) and hexadecylamine (HDA) solutions in various alkanols were investigated by surface tension measurements and ATR-IR spectroscopy. 3D printing experiments also assessed the robustness of the formed films. FINDINGS: Arrested fatty alcohol/water interfaces were formed due to the interfacial association of NaPSS, HDA, and alkanol molecules, which also act as cosurfactants in the surface region. These solid films enable the synthesis of temperature-sensitive all-in-liquid constructs and offer alternatives to bulk polyion/mixed surfactant assemblies prepared earlier through numerous synthesis steps.

Stimuli-Responsive Membrane Anchor Peptide Nanofoils for Tunable Membrane Association and Lipid Bilayer Fusion.

Self-assembled peptide nanostructures with stimuli-responsive features are promising as functional materials. Despite extensive research efforts, water-soluble supramolecular constructs that can interact with lipid membranes in a controllable way are still challenging to achieve. Here, we have employed a short membrane anchor protein motif (GLFD) and coupled it to a spiropyran photoswitch. Under physiological conditions, these conjugates assemble into ∼3.5 nm thick, foil-like peptide bilayer morphologies. Photoisomerization from the closed spiro (SP) form to the open merocyanine (MC) form of the photoswitch triggers rearrangements within the foils. This results in substantial changes in their membrane-binding properties, which also varies sensitively to lipid composition, ranging from reversible nanofoil reformation to stepwise membrane adsorption. The formed peptide layers in the assembly are also able to attach to various liposomes with different surface charges, enabling the fusion of their lipid bilayers. Here, SP-to-MC conversion can be used both to trigger and to modulate the liposome fusion efficiency.

Synthesis of Porous Hollow Organosilica Particles with Tunable Shell Thickness.

Porous hollow silica particles possess promising applications in many fields, ranging from drug delivery to catalysis. From the synthesis perspective, the most challenging parameters are the monodispersity of the size distribution and the thickness and porosity of the shell of the particles. This paper demonstrates a facile two-pot approach to prepare monodisperse porous-hollow silica particles with uniform spherical shape and well-tuned shell thickness. In this method, a series of porous-hollow inorganic and organic-inorganic core-shell silica particles were synthesized via hydrolysis and condensation of 1,2-bis(triethoxysilyl) ethane (BTEE) and tetraethyl orthosilicate (TEOS) in the presence of hexadecyltrimethylammonium bromide (CTAB) as a structure-directing agent on solid silica spheres as core templates. Finally, the core templates were removed via hydrothermal treatment under alkaline conditions. Transmission electron microscopy (TEM) was used to characterize the particles' morphology and size distribution, while the changes in the chemical composition during synthesis were followed by Fourier-transform infrared spectroscopy. Single-particle inductively coupled plasma mass spectrometry (spICP-MS) was applied to assess the monodispersity of the hollow particles prepared with different reaction parameters. We found that the presence of BTEE is key to obtaining a well-defined shell structure, and the increase in the concentration of the precursor and the surfactant increases the thickness of the shell. TEM and spICP-MS measurements revealed that fused particles are also formed under suboptimal reaction parameters, causing the broadening of the size distribution, which can be preceded by using appropriate concentrations of BTEE, CTAB, and ammonia.

Amino Surface Modification and Fluorescent Labelling of Porous Hollow Organosilica Particles: Optimization and Characterization.

Surface modification of silica nanoparticles with organic functional groups while maintaining colloidal stability remains a synthetic challenge. This work aimed to prepare highly dispersed porous hollow organosilica particles (pHOPs) with amino surface modification. The amino-surface modification of pHOPs was carried out with 3-aminopropyl(diethoxy)methylsilane (APDEMS) under various reaction parameters, and the optimal pHOP-NH2 sample was selected and labelled with fluorescein isothiocyanate (FITC) to achieve fluorescent pHOPs (F-HOPs). The prepared pHOPs were thoroughly characterized by transmission electron microscopy, dynamic light scattering, FT-IR, UV-Vis and fluorescence spectroscopies, and microfluidic resistive pulse sensing. The optimal amino surface modification of pHOPs with APDEMS was at pH 10.2, at 60 °C temperature with 10 min reaction time. The positive Zeta potential of pHOP-NH2 in an acidic environment and the appearance of vibrations characteristic to the surface amino groups on the FT-IR spectra prove the successful surface modification. A red-shift in the absorbance spectrum and the appearance of bands characteristic to secondary amines in the FTIR spectrum of F-HOP confirmed the covalent attachment of FITC to pHOP-NH2. This study provides a step-by-step synthetic optimization and characterization of fluorescently labelled organosilica particles to enhance their optical properties and extend their applications.

Hidden behind the mask: An authentication study on the Aztec mask of the Museum of Ethnography, Budapest, Hungary.

Turquoise covered mosaic objects - especially masks - were attractive components of treasures transported to Europe from Mexico after the fall of the Aztec Empire in the 1500s. According to our present knowledge, the mosaic masks were manufactured for ritual purpose. The main material of mosaics, the turquoise was a high-prestige semi-precious stone among Mexican native people. During the 20th century, such objects derived both from illegal treasure hunting and documented archaeological excavations. The aim of our research was the authentication of a turquoise covered Aztec wooden mask, which presumably originates from the Tehuacán Valley, Mexico and exchanged by the Museum of Ethnography, Budapest, in 1973. The detailed and complex analytical investigation of the mask is a curiosity. To reveal the origin of the object, UV photographs were taken, the wooden base was subjected to biological studies and C-14 dating, the organic glue fixing the tesserae and the inorganic mosaic tesserae were investigated by non-destructive chemical, FT-IR and Raman spectroscopic methods. Our investigations determined that the mask of the Museum of Ethnography was made of an alder species of tree and its age is AD 1492-1653. The light-coloured covering mosaic lamellae were identified as alabaster and claystone. Comparing the turquoise tesserae cover with reference materials, their chemical composition has been clearly differentiated from most of the well-known turquoise sources of the US Southwest. Based on our results, the Aztec mask of the Museum of Ethnography proved to be an original piece of art from the 15th-17th century.

Advancement of Fluorescent and Structural Properties of Bovine Serum Albumin-Gold Bioconjugates in Normal and Heavy Water with pH Conditioning and Ageing.

The red-emitting fluorescent properties of bovine serum albumin (BSA)-gold conjugates are commonly attributed to gold nanoclusters formed by metallic and ionized gold atoms, stabilized by the protein. Others argue that red fluorescence originates from gold cation-protein complexes instead, not gold nanoclusters. Our fluorescence and infrared spectroscopy, neutron, and X-ray small-angle scattering measurements show that the fluorescence and structural behavior of BSA-Au conjugates are different in normal and heavy water, strengthening the argument for the existence of loose ionic gold-protein complexes. The quantum yield for red-emitting luminescence is higher in heavy water (3.5%) than normal water (2.4%), emphasizing the impact of hydration effects. Changes in red luminescence are associated with the perturbations of BSA conformations and alterations to interatomic gold-sulfur and gold-oxygen interactions. The relative alignment of domains I and II, II and III, III and IV of BSA, determined from small-angle scattering measurements, indicate a loose ("expanded-like") structure at pH 12 (pD ~12); by contrast, at pH 7 (pD ~7), a more regular formation appears with an increased distance between the I and II domains, suggesting the localization of gold atoms in these regions.

Storage conditions determine the characteristics of red blood cell derived extracellular vesicles.

Extracellular vesicles (EVs) are released during the storage of red blood cell (RBC) concentrates and might play adverse or beneficial roles throughout the utilization of blood products (transfusion). Knowledge of EV release associated factors and mechanism amends blood product management. In the present work the impact of storage time and medium (blood preserving additive vs isotonic phosphate buffer) on the composition, size, and concentration of EVs was studied using attenuated total reflection infrared (ATR-IR) spectroscopy, microfluidic resistive pulse sensing (MRPS) and freeze-fraction combined transmission electron micrography (FF-TEM). The spectroscopic protein-to-lipid ratio based on amide and the C-H stretching band intensity ratio indicated the formation of various vesicle subpopulations depending on storage conditions. After short storage, nanoparticles with high relative protein content were detected. Spectral analysis also suggested differences in lipid and protein composition, too. The fingerprint region (from 1300 to 1000 cm-1) of the IR spectra furnishes additional information about the biomolecular composition of RBC-derived EVs (REVs) such as adenosine triphosphate (ATP), lactose, glucose, and oxidized hemoglobin. The difference between the vesicle subpopulations reveals the complexity of the REV formation mechanism. IR spectroscopy, as a quick, cost-effective, and label-free technique provides valuable novel biochemical insight and might be used complementary to traditional omics approaches on EVs.

Comparison of the Morphological and Structural Characteristic of Bioresorbable and Biocompatible Hydroxyapatite-Loaded Biopolymer Composites.

Calcium phosphate (CaP)-based ceramic-biopolymer composites can be regarded as innovative bioresorbable coatings for load-bearing implants that can promote the osseointegration process. The carbonated hydroxyapatite (cHAp) phase is the most suitable CaP form, since it has the highest similarity to the mineral phase in human bones. In this paper, we investigated the effect of wet chemical preparation parameters on the formation of different CaP phases and compared their morphological and structural characteristics. The results revealed that the shape and crystallinity of CaP particles were strongly dependent on the post-treatment methods, such as heat or alkaline treatment of as-precipitated powders. In the next step, the optimised cHAp particles have been embedded into two types of biopolymers, such as polyvinyl pyrrolidone (PVP) and cellulose acetate (CA). The pure polymer fibres and the cHAp-biopolymer composites were produced using a novel electrospinning technique. The SEM images showed the differences between the morphology and network of CA and PVP fibres as well as proved the successful attachment of cHAp particles. In both cases, the fibres were partially covered with cHAp clusters. The SEM measurements on samples after one week of immersion in PBS solution evidenced the biodegradability of the cHAp-biopolymer composites.

Interplay between membrane active host defense peptides and heme modulates their assemblies and in vitro activity.

In the emerging era of antimicrobial resistance, the susceptibility to co-infections of patients suffering from either acquired or inherited hemolytic disorders can lead to dramatic increase in mortality rates. Closely related, heme liberated during hemolysis is one of the major sources of iron, which is vital for both host and invading microorganisms. While recent intensive research in the field has demonstrated that heme exerts diverse local effects including impairment of immune cells functions, it is almost completely unknown how it may compromise key molecules of our innate immune system, such as antimicrobial host defense peptides (HDPs). Since HDPs hold great promise as natural therapeutic agents against antibiotic-resistant microbes, understanding the effects that may modulate their action in microbial infection is crucial. Here we explore how hemin can interact directly with selected HDPs and influence their structure and membrane activity. It is revealed that induced helical folding, large assembly formation, and altered membrane activity is promoted by hemin. However, these effects showed variations depending mainly on peptide selectivity toward charged lipids, and the affinity of the peptide and hemin to lipid bilayers. Hemin-peptide complexes are sought to form semi-folded co-assemblies, which are present even with model membranes resembling mammalian or bacterial lipid compositions. In vitro cell-based toxicity assays supported that toxic effects of HDPs could be attenuated due to their assembly formation. These results are in line with our previous findings on peptide-lipid-small molecule systems suggesting that small molecules present in the complex in vivo milieu can regulate HDP function. Inversely, diverse effects of endogenous compounds could also be manipulated by HDPs.

Silica@zirconia Core@shell Nanoparticles for Nucleic Acid Building Block Sorption.

The development of delivery systems for the immobilization of nucleic acid cargo molecules is of prime importance due to the need for safe administration of DNA or RNA type of antigens and adjuvants in vaccines. Nanoparticles (NP) in the size range of 20-200 nm have attractive properties as vaccine carriers because they achieve passive targeting of immune cells and can enhance the immune response of a weakly immunogenic antigen via their size. We prepared high capacity 50 nm diameter silica@zirconia NPs with monoclinic/cubic zirconia shell by a green, cheap and up-scalable sol-gel method. We studied the behavior of the particles upon water dialysis and found that the ageing of the zirconia shell is a major determinant of the colloidal stability after transfer into the water due to physisorption of the zirconia starting material on the surface. We determined the optimum conditions for adsorption of DNA building blocks, deoxynucleoside monophosphates (dNMP), the colloidal stability of the resulting NPs and its time dependence. The ligand adsorption was favored by acidic pH, while colloidal stability required neutral-alkaline pH; thus, the optimal pH for the preparation of nucleic acid-modified particles is between 7.0-7.5. The developed silica@zirconia NPs bind as high as 207 mg dNMPs on 1 g of nanocarrier at neutral-physiological pH while maintaining good colloidal stability. We studied the influence of biological buffers and found that while phosphate buffers decrease the loading dramatically, other commonly used buffers, such as HEPES, are compatible with the nanoplatform. We propose the prepared silica@zirconia NPs as promising carriers for nucleic acid-type drug cargos.

Surface enhanced Raman spectroscopic (SERS) behavior of phenylpyruvates used in heterogeneous catalytic asymmetric cascade reaction.

The strength and geometry of adsorption of substituted phenylpyruvates on silver surface was studied by means of surface enhanced Raman spectroscopy (SERS) using silver sol. 2'-nitrophenylpyruvates were used as starting materials in a newly developed heterogeneous catalytic asymmetric cascade reaction to produce substituted quinoline derivatives. Substituents on the aromatic ring of the starting materials had significant influence on the yield of the desired quinoline derivatives. Product selectivity of the transformation of nitrophenylpyruvates were enhanced by the acid added. The geometry and the strength of the adsorption are assumed to play an important role in the outcome of this reaction, so we have tried to find correlation between the structure of adsorbed phenylpyruvates and their catalytic performance. Based on the results of our spectroscopic measurements, the enol form is predominant in the series of phenylpyruvates in solid form and methanol solutions. Stronger adsorption of phenylpyruvates in acidic media through oxygen atoms was indicated by the increased enhancement in the SERS spectrum. The nitro group of 2'-nitrophenylpyruvates has no direct role in the adsorption on Ag surface. This observation has explained why the hydrogenation of the keto group (presumably via the enol form) occurs preferentially and why the formation of the undesired indole derivatives requiring reduction of the nitro group is suppressed. The SERS behavior has helped to get a closer look on the first step of adsorption of starting materials contributing to a better understanding of the cascade reaction studied, thus providing a better flexibility in catalyst design.

Effect of the Microstructure of the Semiconductor Support on the Photocatalytic Performance of the Pt-PtOx/TiO2 Catalyst System.

The influence of the semiconductor microstructure on the photocatalytic behavior of Pt-PtOx/TiO2 catalysts was studied by comparing the methanol-reforming performance of systems based on commercial P25 or TiO2 from sol-gel synthesis calcined at different temperatures. The Pt co-catalyst was deposited by incipient wetness and formed either by calcination or high-temperature H2 treatment. Structural features of the photocatalysts were established by X-ray powder diffraction (XRD), electron spin resonance (ESR), X-ray photoelectron spectroscopy (XPS), optical absorption, Raman spectroscopy and TEM measurements. In situ reduction of Pt during the photocatalytic reaction was generally observed. The P25-based samples showed the best H2 production, while the activity of all sol-gel-based samples was similar in spite of the varying microstructures resulting from the different preparation conditions. Accordingly, the sol-gel-based TiO2 has a fundamental structural feature interfering with its photocatalytic performance, which could not be improved by annealing in the 400-500 °C range even by scarifying specific surface area at higher temperatures.

Construction and Properties of Donor-Acceptor Stenhouse Adducts on Gold Surfaces.

The construction of a donor-acceptor Stenhouse adduct molecular layer on a gold surface is presented. To avoid the incompatibility of the thiol surface-binding group with the donor-acceptor polyene structure of the switch, an interfacial reaction approach was followed. Poly(dopamine)-supported gold nanoparticles on quartz slides were chosen as substrates, which was expected to facilitate both the interfacial reaction and the switching process by providing favorable steric conditions due to the curved particle surface. The reaction between the surface-bound donor half and the CF3-isoxazolone-based acceptor half was proved to be successful by X-ray photoelectron spectroscopy (XPS). However, UV-vis measurements suggested that a closed, cyclopentenone-containing structure of the switch formed on the surface irreversibly. Analysis of the wetting behavior of the surface revealed spontaneous water spreading that could be associated with conformational changes of the closed isomer.

Tamoxifen Delivery System Based on PEGylated Magnetic MCM-41 Silica.

Magnetic iron oxide containing MCM-41 silica (MM) with ~300 nm particle size was developed. The MM material before or after template removal was modified with NH2- or COOH-groups and then grafted with PEG chains. The anticancer drug tamoxifen was loaded into the organic groups' modified and PEGylated nanoparticles by an incipient wetness impregnation procedure. The amount of loaded drug and the release properties depend on whether modification of the nanoparticles was performed before or after the template removal step. The parent and drug-loaded samples were characterized by XRD, N2 physisorption, thermal gravimetric analysis, and ATR FT-IR spectroscopy. ATR FT-IR spectroscopic data and density functional theory (DFT) calculations supported the interaction between the mesoporous silica surface and tamoxifen molecules and pointed out that the drug molecule interacts more strongly with the silicate surface terminated by silanol groups than with the surface modified with carboxyl groups. A sustained tamoxifen release profile was obtained by an in vitro experiment at pH = 7.0 for the PEGylated formulation modified by COOH groups after the template removal. Free drug and formulated tamoxifen samples were further investigated for antiproliferative activity against MCF-7 cells.

Electromagnetic Piezoelectric Acoustic Sensor Detection of Extracellular Vesicles through Interaction with Detached Vesicle Proteins.

An electromagnetic piezoelectric acoustic sensor (EMPAS) was used to study the non-specific adsorption of human red blood cell-derived extracellular vesicle preparations. Vesicle storage history (temperature and duration) highly affected the obtained results: The signal change, namely the frequency decrease of the crystal measured at 20 °C, was negligibly small (<1 s-2) when the vesicle solutions had previously been stored at 4 °C, and was in the order of 10 s-2 when the vesicle solutions had been stored at -30 °C. Moreover, the rate of frequency decrease increased exponentially with the storage time at -30 °C. Upon a 4 °C storage period following the -30 °C storage period of the same sample, the measured frequency decrease dropped, suggesting a partial relaxation of the system. The results are explained by the disintegration of the vesicles triggered by the freeze-thaw cycle, likely due to the detachment of proteins from the vesicle surface as was proved by size-exclusion chromatography. Surface modification of the sensor crystal provided the possibility of signal enhancement, as the maximum rate of the frequency change for the same vesicle concentrations was higher on hydrophobic, octadecyl trichlorosilane-modified quartz than on hydrophilic, bare quartz. The EMPAS signal has been associated with the amount of detached proteins, which in turn is proportional to the originating vesicle concentration.